RESUMEN
This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Técnicas de Reprogramación Celular/métodos , Reprogramación Celular , Vectores Genéticos/genética , Células Madre Pluripotentes Inducidas/citología , Leucocitos Mononucleares/citología , Virus Sendai/genética , Animales , Separación Celular/métodos , Células Cultivadas , Medios de Cultivo , Citocinas/farmacología , Eritroblastos/citología , Eritroblastos/efectos de los fármacos , Eritroblastos/virología , Fibroblastos/citología , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/fisiología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Recombinantes/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/fisiologíaRESUMEN
The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.